Functional RNA molecules such as aptamers, siRNAs, miRNAs, and related compounds have enormous potential as human therapeutics and as tools for elucidating gene regulation in vivo. To reach this potential, such molecules must be highly potent and highly nuclease resistant. Unmodified RNAs typically do not meet these requirements. A variety of chemical modifications have been explored to overcome these limitations. In particular, some success has been achieved in various systems using 2'-O-methyl-ribose and phosphorothioate backbone modifications, alone or in combination. However, further improvements are highly desirable. In addition, phosphorothioate modifications are chiral, resulting in two distinct isomers at each backbone substitution. Thus, there is a need for improved chemical modifications that can be incorporated into functional RNAs. AM Biotechnologies will address these critical issues by developing 2'-O-methyl-ribonucleoside thiophosphoramidites (2'-OMe-thioamidites) to enable synthesis of phosphorodithioate 2'-OMe-RNA (PS2-2'- OMe-RNA). We have previously shown that PS2 modifications at selected backbone positions of DNA aptamers enhance binding affinity to target proteins without loss of specificity. Similarly, selected PS2 modifications in siRNAs significantly improve gene silencing activities. Thus, selected PS2-2'-OMe-RNA modifications will significantly increase binding affinity and potency of 2'-OMe-RNA aptamers, and will offer new avenues for synthesis of highly potent siRNAs. PS2-2'-OMe-RNAs will also be achiral at phosphorus, eliminating the variable biochemical, biophysical, and biological properties of diastereomeric phosphorothioate substituted RNAs. This Phase I project will: 1) develop the chemistry to produce four 2'-OMe-thioamidites (ABz, CBz, GIbu and U);2) optimize the synthesis of PS2-2'-OMe-RNAs;3) evaluate the effects of PS2-2'-OMe modifications on the binding affinity of a model RNA aptamer;and 4) evaluate the effects of PS2-2'-OMe modifications on the gene silencing activities of siRNAs targeting 2-secretase. In Phase II, AM will (a) scale reagent production up to commercial quantities and purity;(b) optimize a robust protocol for synthesis of PS2-2'OMe-RNA;(c) evaluate the effects of PS2-2'-OMe modifications on aptamers and siRNA activity in vivo;and (d) fully characterize the pharmacokinetic properties of PS2-2'-OMe-RNA. AM in Phase II may also offer for sale limited quantities of research-grade reagents for market beta testing. Upon successful completion of Phase II, AM will work with its existing industry partners to commercialize the 2'-OMe-thioamidites and enable the entire life science community to use these unique reagents in developing improved high-potency RNA drugs for a wide variety of human disease applications. PUBLIC HEALTH RELEVANCE: Functional RNA molecules such as aptamers and siRNAs have exciting potential as therapeutics for viral infections, cancer, genetic disorders, and neurological diseases. However, these potential RNA drugs require chemical modifications to achieve the necessary potency and stability. AM Biotechnologies (AM) will develop 2'-O-methyl-ribonucleoside thiophosphoramidite reagents that will allow the life science community to produce high potency, highly stable phosphorodithioate 2'-O-methyl-RNA-based drugs. The unique reagents that AM will develop under this project could have a profound impact on public health.